Site description, sample collection and physico-chemical analysis
Thar Desert also known as Great Indian Desert covers about 2.34 million km² area within India and mainly Bikaner, Barmer, Jaisalmer and Jodhpur districts of Rajasthan encompassing 61% boundary of Thar Desert. It is a large arid region dominated by fixed and mobile sand dunes. The sand dune samples were collected from Bajrang Dhora (28^°03’18.41″ N 73^°15’58″ E) which is located almost 11 km away from Bikaner (Rajasthan), India (Fig. 1). Samplings were carried out in the month June, 2014 and February 2015. Sand dune samples were collected from 10 cm depth of dune surface with the help of sterile spatula and transferred in to sterilized zip lock polythene bags and transported aseptically to laboratory. Physico-chemical conditions (temperature, pH, conductivity, total dissolve solids and salinity) of the soil during sampling were determined at the sampling site by multi-parameter PCSTestr^TM35 (Eutech instrument, Oakton, Singapore).

Isolation and purification of bacterial isolates
Serial dilution method was used for bacterial isolation. Sandy soil sample (1 g) was suspended in 100 mL of sterilized distilled water, slightly homogenized and spreaded onto nutrient agar plates. The plates were incubated at 55 ºC for 24 h¹³. The colonies grown on the plates were purified by successive streaking on nutrient agar plates. Isolated bacterial strains were then subsequently stored at 4 ºC in refrigerator for further study.

Determination of optimum temperature and pH for growth
The physiological growth studies were carried out in the nutrient broth medium as described previously ¹⁷. The temperature range for optimum growth was determined by incubating the isolates from 25 to 60 ºC. The effect of pH on growth of isolates was tested with the pH range from 4.0 to 10.0. All growth tests were done at 50 ºC for 24 h and the growth was measured at OD₆₀₀ nm by using spectrophotometer (HALO DB-30 UV-Visible double beam, Dynamica, Singapore).

Morphological studies of colony and cell
The appearance of colony such as shape, colour, opacity, margin and elevation was observed by magnifying lens (10X) after the growth of isolated strains on nutrient agar plates. The cell size was measured using ocular micrometer. Gram staining was performed using standard¹⁸. The various morphological features of bacterial strains were observed at 1000X using Axio Lab.A1 compound microscope (Carl Zeiss, Germany).

Biochemical characterization
A series of biochemical tests were performed at 50 ºC for 24 h at pH 7.0 using standard protocols¹⁹. Briefly, catalase activity was determined by adding a 3 % (w/v) H₂O₂ solution on colonies of isolates. Oxidase test was done by placing a loopful bacterial isolate on oxidase disc (Himedia, India) and the change in color was observed. Indole test was performed in tryptone broth after adding 0.5 ml Kovac’s reagent and waited for color change. Methyl Red (MR) and Voges-Proskauer (VP) tests were performed for acid production ability of strains. Utilization of citrate by isolates was observed on Simmons citrate agar (Himedia, India) medium slants. Urease enzyme production from urea was determined with suspension of bacterial strains inoculated in Christensen’s urea broth (CUB). Reduction of nitrate was determined in nitrate broth medium. Fermentation of carbohydrates was performed using triple sugar iron agar medium.

Screening of isolates for extracellular hydrolytic activities
Extracellular protease production
The qualitative screening of protease enzyme production by isolates was achieved using skim milk in nutrient agar medium with following components (g l^-1): skim milk, 10; peptic digest of animal tissue, 5; sodium chloride, 5; beef extract, 1.5; yeast extract, 1.5; and agar, 15 and pH adjusted to 7.0 before autoclaving. Isolates were streaked on medium plates. A zone of hydrolysis around the colonies was considered as an evidence of proteolytic activity ²⁰.

Extracellular amylase production
The amylase production was determined by growing the isolates on soluble starch containing nutrient agar medium with the following constituents (g l^-1): soluble starch, 10; peptic digest of animal tissue, 5; sodium chloride, 5; beef extract, 1.5; yeast extract, 1.5; and agar, 15 (pH adjusted to 7.0 before autoclaving). Isolates were streaked in straight lines on medium plates. After incubation, individual plates were flooded with Gram’s iodine solution (3% (w/v), KI and 0.3% (w/v) iodine). A clear zone around the growth indicated hydrolysis of starch²¹.

Extracellular lipase production
Lipase activity of the isolates was detected by screening for turbid halo or zones of precipitation around colonies growing on nutrient agar plates containing 1% Tween-80²².

Extracellular asparaginase production
The ability of isolates to produce asparaginase was determined using nutrient agar medium with L-asparagine. The composition of the test medium was as follows (g l^-1): L-asparagine, 20; glucose, 2; potassium chloride, 0.5; magnesium sulfate heptahydrate, 0.5; monopotassium phosphate, 1.52; agar, 20 and phenol red, 0.09 and pH 7.0. Clear pink zone around the colonies after the incubation of petri plates indicated the asparaginase production²³.

Extracellular inulinase production
The production of inulinase enzyme by isolates was screened using inulin agar medium plates containing following constituents (g l^-1): ammonium sulfate, 0.5; magnesium sulfate heptahydrate, 0.2; inulin, 2; monopotassium phosphate, 3; agar, 20 and pH 7.0. After incubation, plates were flooded with Lugol’s iodine solution for 3 to 5 minutes and washed with distilled water.  The colorless zone around the colonies indicated inulinase enzyme production²⁴.

Extracellular cellulase production
Cellulolytic activity of isolates was detected on carboxymethylcellulose (CMC) agar plates. Isolates were streaked on CMC agar medium plates and incubated at 50 °C for 24 h to allow the secretion of cellulase. After the incubation, plates were flooded with congo red (1% w/v) for 15 min. Subsequently, congo red solution was discarded and 1M NaCl solution added on plates for 15 min. The hydrolysis zone around the colonies indicated the cellulolytic activity ⁶.

Genomic DNA extraction
Genomic DNA of isolates was extracted using modified freeze/thaw method²⁵. Briefly, bacterial culture was pelleted out at 9,000 g for 5 min and lysis solution (500 µl) added (mixed gently by inverting 3-6 times) and incubated at -20 °C for 15 minutes. Later, tubes were incubated at 70 °C for 15 min in water bath and this step was repeated. Cells were suspended in 600 µl of Chloroform and gently emulsified by inversing tubes for 3-5 times and centrifuged at 9,000 g for 3 min. Upper aqueous phase containing DNA was transferred to a new tube and equal amount of Propan-2-OL (iso-propyl alcohol) was added and mixed gently by inversion at room temperature and centrifuged at 9,000 g for 3 min. Supernatant was removed completely and DNA pellet was dissolved in 100 µl of 1.2 M NaCl solution. 300 µl of absolute cold ethanol added and the DNA was precipitated in 10 min at -20 °C. Later, the tubes were centrifuged at 9,000 g for 5 min. Ethanol was removed and pellet washed once with 70% cold ethanol and tubes were centrifuged at 9,000 g for 5 min. Lastly, the pellet was dissolved in 50 µl deionized water (pH 7.0) by gentle vortexing and kept at -20 °C for further use.

16S rRNA gene Sequencing and phylogenetic analysis
The 16S rRNA gene of genomic DNA was amplified using PCR master mix (Genei^TM Company) with the universal primers 27F and 1495R²⁶. The PCR thermal cycling conditions were as follows: Hot start at 98 °C for 5 min, followed by 35 cycles denaturation at 95 °C for 30 s, primer annealing at 56 °C for 45 s, elongation at 72 °C for 1.45 min with final extension at 72 °C for 5 min. Amplified genomic DNA was purified with GeneJET PCR purification kit (Thermo Fisher) as per the manufacturer’s protocol. Purified products were sequenced commercially (Eurofins Genomics India, Bangalore) to get partial sequences 16S rRNA gene.

The similarity search of 16S rRNA gene sequences of isolated strains was done through the Eztaxon database²⁷ and parallel search also performed using NCBI database using BLASTN (www.ncbi.nlm.nih.gov/blastn) to retrieve closely related sequences for phylogenetic analysis. Initially, a large group of sequences (>1100 bp) was selected and later a smaller subset was employed for analysis based on the relatedness of the sequences and excluding taxa of uncertain affiliation. The multiple sequences alignment was performed using programme MUSCLE²⁸ inbuilt in the phylogenetic analysis software MEGA 7²⁹. The aligned sequences were further refined manually to obtain unambiguous data. Finally, the sequences of type strains and other bacterial strains mostly allied to sequences of the present study were employed for a synoptic view of the phylogenetic tree. The phylogenetic tree using 16S rRNA gene fragment of 1117 nucleotides was constructed by the maximum likelihood (ML) method using the program MEGA 7²⁹ applying a T92+G model of nucleotide substitution, which gave the best fit to this dataset according to the Bayesian Information Criterion (BIC). Bootstrapping of 1000 replications was calculated to find out confidence values for the edges of the maximum likelihood tree. Planococcus citreus (X62172) was chosen as the outgroup.

Nucleotide sequence accession numbers
The nucleotide sequences determined in this study have been deposited in the GenBank (NCBI database) under accession numbers MH560567-MH560574.